The field of the present invention is the area of DNA repair enzymes. In particular, the invention concerns the identification of stable ultraviolet DNA endonuclease polypeptide fragments, their nucleotide sequences and recombinant host cells and methods for producing them and for using them in DNA repair processes.
The integrity of its genetic material must be maintained in order for a biological species to survive. However, DNA is continuously subject to damage by endogenous and exogenous agents that can lead to mutations, neoplasia or cell death [Smith et al. (1996) Biochemistry 35:4146–4154; Brash et al. (1991) Proc. Natl. Acad. Sci. USA 88:10124–10128]. One potential source of mutations is nucleotide misincorporation by DNA polymerases during DNA replication or repair. In addition, primer/template slippage can occur at repetitive DNA sequences during replication, resulting in single-stranded loops of one or more unpaired bases called insertion/deletion loops (IDLs) that can be mutagenic [Sancar, A. (1999) Nat. Genet. 21:247–249]. The human genome has an abundance of simple repeat sequences that are relatively unstable [Petruska et al. (1998) J. Biol. Chem. 273(9):5204–5210]. Expansion of such repeat sequences have been associated with human genetic diseases including Huntington's disease, fragile X syndrome and myotonic dystrophy [Pearson et al. (1998) Nucleic Acids Res. 26(3):816–823].
The Escherichia coli Mut HLS pathway has been extensively characterized and is the prototypical DNA mismatch repair (MMR) pathway. This repair pathway recognizes and repairs small IDLs and all single base mismatches except C/C in a strand-specific manner [Modrich, P. (1991) Annu. Rev. Genet. 25:229–253]. Mismatch repair pathways have been highly conserved during evolution [Modrich and Lahaue (1996) Annu. Rev. Biochem. 65:101–133]. Eukaryotes including Saccharomyces cerevisiae and humans have several genes encoding proteins homologous to bacterial MutL and MutS [Sancar, A. (1999) supra]. For example, there are six MutS (Msh1–Msh6) and four MutL (MLH1–3, PMS1) homologs in S. cerevisiae [Kolodoner, R. (1996) Genes Dev. 10:1433–1442]. The Msh2p–Msh6p heterodimer binds base mismatches and small IDLs whereas the Msh2p–Msh3p heterodimer binds only small and large IDLs [Marsischky et al. (1996) Genes Dev. 10(4):407–420]. A considerable amount of evidence implicates mismatch repair in stabilizing repetitive DNA sequences [Marsischky et al. (1996) supra; Fujii et al. (1999) J. Mol. Biol. 289:835–850; Strand et al. (1993) Nature 365:274–276].
Cellular exposure to ultraviolet radiation (UV) results in numerous detrimental effects including cell death, mutation and neoplastic transformation. Studies indicate that some of these deleterious effects are due to the formation of two major classes of bipyrimidine DNA photoproducts, cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4 PPs). [Friedberg et al. (1995) In: DNA Repair and Mutagenesis, Am. Soc. Microbiol., Washington, D.C., pp. 24–31].
Organisms have evolved several different pathways for removing CPDs and 6-4 PPs from cellular DNA [Friedberg et al. (1995) supra; Brash et al. (1991) supra]. These pathways include direct reversal and various excision repair pathways which can be highly specific or nonspecific for CPDs and 6-4 PPs. For example, DNA photolyases specific for either CPDs or 6-4 PPs have been found in a variety of species and restore the photoproduct bases back to their original undamaged states [Rubert, C. S. (1975) Basic Life Sci. 5A:73–87; Kim et al. (1994) J. Biol. Chem. 269:8535–8540; Sancar, G. B. (1990) Mutat. Res. 236:147–160]. Excision repair has been traditionally divided into either base excision repair (BER) or nucleotide excision repair (NER) pathways, which are mediated by separate sets of proteins but which both are comprised of DNA incision, lesion removal, gap-filling and ligation reactions [Sancar, A. (1994) Science 266:1954–19560; Sancar and Tang (1993) Photochem. Photobiol. 57:905–921]. BER N-glycosylase/AP lyases specific for CPDs cleave the N-glycosidic bond of the CPD 5′ pyrimidine and then cleave the phosphodiester backbone at the abasic site via a β-lyase mechanism, and have been found in several species including T4 phage-infected Escherichia coli, Micrococcus luteus, and Saccharomyces cerevisiae [Nakabeppu et al. (1982) J. Biol. Chem. 257:2556–2562; Grafstrom et al. (1982) J. Biol. Chem. 257:13465–13474; Hamilton et al. (1992) Nature 356:725–728]. NER is a widely distributed, lesion non-specific repair pathway which orchestrates DNA damage removal via a dual incision reaction upstream and downstream from the damage site, releasing an oligonucleotide containing the damage and subsequent gap filling and ligation reactions [Sancar and Tang (1993) supra].
Recently, an alternative excision repair pathway initiated by a direct acting nuclease which recognizes and cleaves DNA containing CPDs or 6-4 PPs immediately 5′ to the photoproduct site has been described [Bowman et al. (1994) Nucleic. Acids Res. 22:3026–3032; Freyer et al. (1995) Mol. Cell. Biol. 15:4572–4577; Doetsch, P. W. (1995) Trends Biochem. Sci. 20:384–386; Davey et al. (1997) Nucleic Acids Res. 25:1002–1008; Yajima et al. (1995) EMBO J. 14:2393–2399; Yonemasu et al. (1997) Nucleic Acids Res. 25:1553–1558; Takao et al. (1996) Nucleic Acids Res. 24:1267–1271]. The initiating enzyme has been termed UV damage endonuclease (UVDE, now termed Uve1p). Homologs of UVDE have been found in Schizosaccharomyces pombe, Neurospora crassa and Bacillus subtilis [Yajima et al. (1995) supra; Yonemasu et al. (1997) supra; Takao et al. (1996) supra]. The Uve1p homologs from these three species have been cloned, sequenced and confer increased UV resistance when introduced into UV-sensitive strains of E. coli, S. cerevisiae, and human cells [Yajima et al. (1995) supra; Takao et al. (1996) supra]. In S. pombe Uve1p is encoded by the uve1+gene. However, because of the apparently unstable nature of partially purified full-length and some truncated UVDE derivatives, UVDE enzymes have been relatively poorly characterized and are of limited use [Takao et al. (1996) supra].
Because of the increasing and widespread incidence of skin cancers throughout the world and due to the reported inherent instability of various types of partially purified full-length and truncated UVDE derivatives, there is a long felt need for the isolation and purification of stable UVDE products, especially for use in skin care and medicinal formulations.